The present disclosure relates to quantitative fluorescence imaging and more specifically to wide-field fluorescence imaging systems and methods. The various embodiments enable accurate, quantitative, fast, contiguous wide-field fluorescence imaging such as laser line scanning with near perfect registration and measurement across the entire field of view.
Recently, there is a growing desire by the scientific research community to include fluorescence detection in tissue imaging tools. Fluorescence detection provides a more controllable, stable way of identifying the impact of certain drugs, for example. A number of automated microscope systems now include fluorescence imaging capabilities. Most of these systems were built by automating the stage of a microscope or adding a microscopic imager to an automated scanner. Their focus has mostly been to automate the tasks that a typical Pathologist performs as he/she inspects a tissue slide under a microscope. This meant that such a system must be a microscope first. Microscopic imaging does provide great benefits, including sub-cellular details and a potential for matching what a Pathologist sees directly through a microscope, but at the same time it tends to be quite slow. A microscope objective images a very small area. For example, a 20× NA=0.75 objective images an area less than 0.5 mm wide at a resolution ˜0.4 μm. So, a slide area of 50 mm×25 mm would require 5000 images with stop-and-go tiled imaging. This is generally not a problem for color imaging, like imaging H&E (Hematoxylin and Eosin) stains, since short exposure times are enough to detect the signal. However, for fluorescence imaging, much longer exposure times would be needed for low abundance labeling and therefore these methods result in much longer scan times. Combining the longer scan times with the fact that a typical experiment requires the scanning of a number of slides in order to determine the area of interest to investigate further means that the total processing cycle per experiment can turn into hours if not days.
Of the faster microscope automation techniques is the technique disclosed in U.S. Pat. No. 8,385,619 developed by Soenksen at Aperio Technologies, Inc., now part of Leica Biosystems. Soenksen recognized the need to speed up the automation of slide imaging in microscope systems and implemented line imaging as a way to reduce the number of images to tile (strips). Soenksen used a line scan Time-Delay-Integration (TDI) camera, along with the objective and focusing optics, to image one line at a time. The TDI camera allows for broad illumination (with a lamp or LED) and reads the image as one line at a time. This technique does improve microscope automation and achieves faster scanning results and less tiling mismatch issues. The achieved scan times seem to be acceptable for direct color imaging (H&E stains) where exposure times per imaged line can be short so that the total time it takes to cover a wider area can be reasonable. But, for fluorescence, this technique still requires longer exposure times per line and the result is much longer total scan times. This presents a bottleneck for the cases where there exists a set of slides to go through before the researcher would know if he/she has what he's looking for or not. Based on the time scanning slides takes alone, there is still a need for a fast triage step to determine which slides have the area(s) that would be worth scanning on an automated microscope system. It is desirable for this triage step to be sensitive so it does not miss what can be detected by systems downstream. Also, equally desirable, is the accuracy of the relative location and relative signal reproduction so that accurate assessment of whether or not to go to next steps in the process and if so where exactly to scan at high resolution.
Microscope based systems such as that disclosed in U.S. Pat. No. 8,385,619 allow for “macroscopic imaging” through the use of a lower magnification objective to image a wider field of view per pass and thus cover a larger area in less time. However, this approach suffers from at least two major limitations. First, it requires much longer exposure time per line image because the NA of low magnification objective is much lower than a high magnification objective. For example, a typical 2× objective has an NA less than 0.075 compared to NA=0.75 for 20× which translates to light collection efficiency ˜(0.75/0.075)^2=100 times smaller. Second, the larger the field of view of a microscope, the more fall-off and distortion there is towards the perimeter of the field of view. This in turn translates into variations in sensitivity across the field of view and inaccurate registration between passes, respectively. These limitations are inherent to the way an objective based imaging system works.
Another key drawback in the existing art for fluorescence imaging, both microscopic and macroscopic, is the variation in signal throughput and optical background suppression across the field of view due to angular spectral shifting of interference filters. The emission spectrum of many fluorescence dyes are narrow and have steep slopes. FIG. 1 shows a typical absorption and emission spectra for LI-COR's IRDye® 800cw. In FIG. 1, a typical long-pass filter that can be used to select a certain window in the emission path is also superposed on the plot. It shows the transmission spectrum of the filter under two incident light conditions: Zero degree angle of incidence (curve 3a) and 20 degree angle of incidence (curve 3b). In most cases for the IRDye® 800cw, the edge for the long-pass filter would need to be placed on the steep slope of the emission curve 2 in order to allow room for excitation light to be matched with the absorption curve 1. This means that if the incidence angle changes when light goes through the filter, the amount of light collected (at that angle) changes. If light collected from different points in the field of view end up going through the filter at different angles, the resultant measurement is not the same even if both locations were illuminated by the same amount of excitation light. Table 1 shows an example of the amount of spectral shifting for a typical interference filter. In many applications this amount is not significant, especially in microscopy applications where there are other more significant factors that may limit its usefulness for quantitative measurements, for example its sensitivity to focus variability. For most microscope systems, including the system disclosed in U.S. Pat. No. 8,385,619, there is no provision to avoid this problem and therefore filters are commonly placed between the objective and the focusing optics where, by definition of imaging by an objective, light from different field points must go through different angles at the side of the objective opposite to the sample side (See, e.g., FIG. 2 of U.S. Pat. No. 8,385,619, element 50 and FIG. 2 of US Patent Application 2011/0121199 to Tanikawa, element 13).
The current inventor realized the need for enhancing background suppression across the whole field of view equally and applied it to imaging small animals. See, e.g., U.S. Pat. No. 7,286,232 . FIG. 2 shows one embodiment of this method wherein a telecentric space 18 is created between the imaging optics 12 and the detector array 13 so that light collected from different points on the target 10 go through the emission filter 15 with the same angular range. A rejection filter 14 was also added between the target area 10 and the imaging optics 12 to further enhance the filtering rejection. The telecentric space is created by placing an aperture 16 at the front focal plane of the imaging optics 12. That worked well for that purpose and any similar single shot macro-imaging. There was no need to worry about scanning the target to cover a larger area as is the case here and therefore there was no concern about the angular variation as light goes through the rejection filter as well. The goal then for the rejection filter was to suppress reflected light as an enhancement to the main emission filter which is placed in the telecentric space. There is no clear way to apply this front aperture technique to the rejection filter without sacrificing signal (i.e. reducing the imaging NA) and that's not desirable for low abundance labels such as in tissue sections and tissue arrays.
Others have recognized the usefulness of telecentric projections to achieve various tasks but not with the functions needed here, namely contiguous, wide-field imaging with spectral filtering uniform across the whole field of view. US patent Application 2012/0313008 to Sung-Ho Jo provides a fluorescence detector design that has a telecentric lens positioned between the fluorescence selecting unit (filter) and the light receiving unit (detector). The purpose of using this telecentric lens is to keep lights collected from different wells separate. Hence, this is not an imaging application where a contiguous area or line is imaged at the same time. Besides, the fluorescence selecting unit is still in non-telectric space. A similar design in U.S. Pat. No. 7,687,260 to Gutekunst is provided for collecting light from an array of sites (wells). Here, the telecentric space is created on the object side by using a field lens on top of the wells. This too does not address the filtering variability. Imaging filter 9 (Guntekunst FIG. 1) is still in non-telecentric space. Furthermore, this is not wide-field, contiguous imaging where stricter requirements on distortion and relative positional accuracy are of concern. This technique is neither applicable to the present wide-field imaging problem by itself nor in combination with other above techniques.
Other telecentric based ideas also exist in flow cytometry where, again, it's not a wide-field imaging application. For example, U.S. Pat. No. 8,467,055 to Imanishi discloses use of a lens 48 to create a telecentric space on the detector array side so that the different beams created by a grating 47 enter the detector sites at similar angles. And again, here, there is no concern and therefore no special provisions for where spectral filters are placed.
Therefore, there is still a need for a more robust, quantitative, fast macroscopic fluorescence imager that does not have the limitations of angular dependence on where in the field the light originates from. Furthermore, there is still a need to accurately maintain the relative locations of the origins of fluorescence light on the sample so that multi-pass images are aligned accurately and thus eliminate the focus dependent positional shifting present in current macroscopic wide-field imagers.